Process for the production of L-lysine
The invention relates to mutants of coryneform bacteria in which genes have been enhanced by the use of a mutated promoter region, and to processes for the production of amino acids using bacteria according to the invention.
1. An isolated mutant coryneform bacterium comprising a DNA fragment with promoter activity operably linked to a polynucleotide coding for a protein with dihydrodipicolinate reductase activity, wherein said DNA fragment comprises the sequence of SEQ ID NO:12 but with one or more substitutions selected from the group consisting of:
a) substitution of the nucleobase thymine at position 36 of SEQ ID NO:12 by guanine;
b) substitution of the nucleobase cytosine at position 37 of SEQ ID NO:12 by guanine;
c) substitution of the nucleobase guanine at position 40 of SEQ ID NO:12 by thymine;
d) substitution of the nucleobase cytosine at position 42 of SEQ ID NO:12 by adenine;
and wherein:
i) relative to a bacterial parent strain which does not carry said DNA fragment operably linked to a polynucleotide coding for a protein with dihydrodipicolinate reductase activity, said mutant coryneform bacterium produces more L-lysine when fermented in nutrient medium and under conditions suitable for lysine production; and
ii) relative to a DNA fragment of SEQ ID NO:12 without any of said substitutions, said DNA fragment with the sequence of SEQ ID NO:12 with one or more of said substitutions has increased transcriptional activity.
2. The isolated mutant coryneform bacterium of claim 1 , wherein, relative to a bacterial parent strain which does not carry said DNA fragment operably linked to a polynucleotide coding for a protein with dihydrodipicolinate reductase activity, said mutant coryneform bacterium produces at least 0.5% more L-lysine when fermented in nutrient medium and under conditions suitable for lysine production.
3. The isolated mutant coryneform bacterium of claim 1 , wherein, relative to a bacterial parent strain which does not carry said DNA fragment operably linked to a polynucleotide coding for a protein with dihydrodipicolinate reductase activity, said mutant coryneform bacterium produces at least 1% more L-lysine when fermented in nutrient medium and under conditions suitable for lysine production.
4. The isolated mutant coryneform bacterium of claim 1 , wherein said DNA fragment comprises at least two of said substitutions.
5. The isolated mutant coryneform bacterium of claim 1 , wherein said DNA fragment comprises at least three of said substitutions.
6. The isolated mutant coryneform bacterium of claim 1 , wherein said DNA fragment comprises all four of said substitutions.
7. The isolated mutant coryneform bacterium of claim 6 , wherein, relative to a bacterial parent strain which does not carry said DNA fragment operably linked to a polynucleotide coding for a protein with dihydrodipicolinate reductase activity, said mutant coryneform bacterium produces at least 0.5% more L-lysine when fermented in nutrient medium and under conditions suitable for lysine production.
8. The isolated mutant coryneform bacterium of claim 7 , wherein said polynucleotide codes for a polypeptide whose amino acid sequence is at least 90% identical to the amino acid sequence of SEQ ID NO:2.
9. The isolated mutant coryneform bacterium of claim 8 , wherein said bacterium is of the species Corynebacterium glutamicum.
10. The isolated mutant coryneform bacterium of claim 9 , wherein, relative to a bacterial parent strain which does not carry said DNA fragment operably linked to a polynucleotide coding for a protein with dihydrodipicolinate reductase activity, said mutant coryneform bacterium produces at least 1% more L-lysine when fermented in nutrient medium and under conditions suitable for lysine production.
11. The isolated mutant coryneform bacterium of claim 10 , wherein, relative to a bacterial parent strain which does not carry said DNA fragment operably linked to a polynucleotide coding for a protein with dihydrodipicolinate reductase activity, said mutant coryneform bacterium produces at least 2% more L-lysine when fermented in nutrient medium and under conditions suitable for lysine production.
12. The isolated mutant coryneform bacterium of claim 11 , wherein said DNA fragment comprises all four of said substitutions and, relative to a bacterial parent strain which does not carry said DNA fragment operably linked to a polynucleotide coding for a protein with dihydrodipicolinate reductase activity, said mutant coryneform bacterium has at least 10% more dihydrodipicolinate reductase activity.
13. An isolated mutant of a coryneform bacterium which comprises a DNA fragment with promoter activity, wherein said DNA fragment has the sequence of SEQ ID NO:12 but with all of the following substitutions:
a) substitution of the nucleobase thymine at position 36 of SEQ ID NO:12 by guanine;
b) substitution of the nucleobase cytosine at position 37 of SEQ ID NO:12 by guanine;
c) substitution of the nucleobase guanine at position 40 of SEQ ID NO:12 by thymine;
d) substitution of the nucleobase cytosine at position 42 of SEQ ID NO:12 by adenine;
and wherein, relative to a DNA fragment comprising the sequence of SEQ ID NO:12 without any of said substitutions, said DNA fragment with said substitutions has increased transcriptional activity.
14. The isolated mutant of a coryneform bacterium of claim 13 , wherein relative to a bacterial parent strain which does not carry a DNA fragment comprising the sequence of SEQ ID NO:12 with said substitutions said isolated mutant coryneform bacterium produces more L-lysine when fermented in nutrient medium and under conditions suitable for lysine production.
15. The isolated mutant coryneform bacterium of claim 14 , wherein said bacterium is of the species Corynebacterium glutamicum.
16. The isolated mutant coryneform bacterium of claim 15 , wherein, relative to a bacterial parent strain which does not carry said DNA fragment with said substitutions, said mutant coryneform bacterium produces at least 0.5% more L-lysine when fermented in nutrient medium and under conditions suitable for lysine production.
17. The isolated mutant coryneform bacterium of claim 15 , wherein, relative to a bacterial parent strain which does not carry said DNA fragment with said substitutions, said mutant coryneform bacterium produces at least 1% more L-lysine when fermented in nutrient medium and under conditions suitable for lysine production.
18. The isolated mutant coryneform bacterium of claim 15 , wherein, relative to a bacterial parent strain which does not carry said DNA fragment with said substitutions, said mutant coryneform bacterium has at least 10% more dihydrodipicolinate reductase activity.
19. The isolated mutant coryneform bacterium of claim 16 , wherein said polynucleotide codes for a polypeptide whose amino acid sequence is at least 90% identical to the amino acid sequence of SEQ ID NO:2.
20. A process for the production of L-lysine comprising:
a) fermenting the isolated mutant coryneform bacterium of claim 13 in a medium to produce a fermentation liquor;
b) allowing L-lysine to accumulate in said fermentation liquor or in said mutant bacterium;
c) after step b), collecting or isolating said L-lysine along with >0 to 100% of biomass and/or fermentation liquor constituents.
21. The process claim 20 , wherein:
a) relative to a bacterial parent strain which does not carry a DNA fragment comprising the sequence of SEQ ID NO:12 with said substitutions, said isolated mutant coryneform bacterium produces more L-lysine when fermented in nutrient medium and under conditions suitable for lysine production;
b) said isolated mutant coryneform bacterium is of the species Corynebacterium glutamicum.
22. The process of claim 21 , wherein, relative to a bacterial parent strain which does not carry said DNA fragment with said substitutions, said isolated mutant coryneform bacterium produces at least 0.5% more L-lysine.
23. The isolated mutant coryneform bacterium of claim 22 , wherein said polynucleotide codes for a polypeptide whose amino acid sequence is at least 90% identical to the amino acid sequence of SEQ ID NO:2.
24. The process of claim 23 , wherein, relative to a bacterial parent strain which does not carry said DNA fragment with said substitutions, said isolated mutant coryneform bacterium produces at least 1% more L-lysine.
25. The process of claim 23 , wherein, after step b), said L-lysine is purified.
26. The process of claim 23 , wherein, after step b), said L lysine together with said fermentation liquor and said biomass are collected.